BRaf signaling principles unveiled by large-scale human mutation analysis with a rapid lentivirus-based gene replacement method.

Rapid advances in genetics are linking mutations on genes to diseases at an exponential rate, yet characterizing the gene-mutation-cell-behavior relationships essential for precision medicine remains a daunting task. More than 350 mutations on small GTPase BRaf are associated with various tumors, and ∼40 mutations are associated with the neurodevelopmental disorder cardio-facio-cutaneous syndrome (CFC). We developed a fast cost-effective lentivirus-based rapid gene replacement method to interrogate the physiopathology of BRaf and ∼50 disease-linked BRaf mutants, including all CFC-linked mutants. Analysis of simultaneous multiple patch-clamp recordings from 6068 pairs of rat neurons with validation in additional mouse and human neurons and multiple learning tests from 1486 rats identified BRaf as the key missing signaling effector in the common synaptic NMDA-R-CaMKII-SynGap-Ras-BRaf-MEK-ERK transduction cascade. Moreover, the analysis creates the original big data unveiling three general features of BRaf signaling. This study establishes the first efficient procedure that permits large-scale functional analysis of human disease-linked mutations essential for precision medicine.

Dysfunction of NMDA receptors in neuronal models of an autism spectrum disorder patient with a DSCAM mutation and in Dscam-knockout mice.

Heterogeneity in the etiopathology of autism spectrum disorders (ASD) limits the development of generic remedies, requires individualistic and patient-specific research. Recent progress in human-induced pluripotent stem cell (iPSC) technology provides a novel platform for modeling ASDs for studying complex neuronal phenotypes. In this study, we generated telencephalic induced neuronal (iN) cells from iPSCs derived from an ASD patient with a heterozygous point mutation in the DSCAM gene. The mRNA of DSCAM and the density of DSCAM in dendrites were significantly decreased in ASD compared to control iN cells. RNA sequencing analysis revealed that several synaptic function-related genes including NMDA receptor subunits were downregulated in ASD iN cells. Moreover, NMDA receptor (R)-mediated currents were significantly reduced in ASD compared to control iN cells. Normal NMDA-R-mediated current levels were rescued by expressing wild-type DSCAM in ASD iN cells, and reduced currents were observed by truncated DSCAM expression in control iN cells. shRNA-mediated DSCAM knockdown in control iN cells resulted in the downregulation of an NMDA-R subunit, which was rescued by the overexpression of shRNA-resistant DSCAM. Furthermore, DSCAM was co-localized with NMDA-R components in the dendritic spines of iN cells whereas their co-localizations were significantly reduced in ASD iN cells. Levels of phospho-ERK1/2 were significantly lower in ASD iN cells, suggesting a potential mechanism. A neural stem cell-specific Dscam heterozygous knockout mouse model, showing deficits in social interaction and social memory with reduced NMDA-R currents. These data suggest that DSCAM mutation causes pathological symptoms of ASD by dysregulating NMDA-R function.

Pharmacological rescue of Ras signaling, GluA1-dependent synaptic plasticity, and learning deficits in a fragile X model.

Fragile X syndrome, caused by the loss of Fmr1 gene function, is the most common form of inherited mental retardation, with no effective treatment. Using a tractable animal model, we investigated mechanisms of action of a few FDA-approved psychoactive drugs that modestly benefit the cognitive performance in fragile X patients. Here we report that compounds activating serotonin (5HT) subtype 2B receptors (5HT2B-Rs) or dopamine (DA) subtype 1-like receptors (D1-Rs) and/or those inhibiting 5HT2A-Rs or D2-Rs moderately enhance Ras-PI3K/PKB signaling input, GluA1-dependent synaptic plasticity, and learning in Fmr1 knockout mice. Unexpectedly, combinations of these 5HT and DA compounds at low doses synergistically stimulate Ras-PI3K/PKB signal transduction and GluA1-dependent synaptic plasticity and remarkably restore normal learning in Fmr1 knockout mice without causing anxiety-related side effects. These findings suggest that properly dosed and combined FDA-approved psychoactive drugs may effectively treat the cognitive impairment associated with fragile X syndrome.

Endoplasmic Reticulum Stress Increases DUSP5 Expression via PERK-CHOP Pathway, Leading to Hepatocyte Death.

Hepatocyte death is critical for the pathogenesis of liver disease progression, which is closely associated with endoplasmic reticulum (ER) stress responses. However, the molecular basis for ER stress-mediated hepatocyte injury remains largely unknown. This study investigated the effect of ER stress on dual-specificity phosphatase 5 (DUSP5) expression and its role in hepatocyte death. Analysis of Gene Expression Omnibus (GEO) database showed that hepatic DUSP5 levels increased in the patients with liver fibrosis, which was verified in mouse models of liver diseases with ER stress. DUSP5 expression was elevated in both fibrotic and acutely injured liver of mice treated with liver toxicants. Treatment of ER stress inducers enhanced DUSP5 expression in hepatocytes, which was validated in vivo condition. The induction of DUSP5 by ER stress was blocked by either treatment with a chemical inhibitor of the protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathway, or knockdown of C/EBP homologous protein (CHOP), whereas it was not affected by the silencing of IRE1 or ATF6. In addition, DUSP5 overexpression decreased extracellular-signal-regulated kinase (ERK) phosphorylation, but increased cleaved caspase-3 levels. Moreover, the reduction of cell viability under ER stress condition was attenuated by DUSP5 knockdown. In conclusion, DUSP5 expression is elevated in hepatocytes by ER stress through the PERK-CHOP pathway, contributing to hepatocyte death possibly through ERK inhibition.

Adenomatous polyposis coli-stimulated GEF 1 (Asef1) is a negative regulator of excitatory synaptic function.

Guanine nucleotide exchange factors (GEFs) play important roles in many cellular processes, including regulation of the structural plasticity of dendritic spines. A GEF protein, adenomatous polyposis coli-stimulated GEF 1 (Asef1, ARHGEF4) is highly expressed in the nervous system. However, the function of Asef1 has not been investigated in neurons. Here, we present evidence showing that Asef1 negatively regulates the synaptic localization of postsynaptic density protein 95 (PSD-95) in the excitatory synapse by inhibiting Staufen-mediated synaptic localization of PSD-95. Accordingly, Asef1 expression impairs synaptic transmission in hippocampal cultured neurons. In addition, neuronal activity facilitates the dissociation of Asef1 from Staufen in a phosphoinositide 3 kinase (PI3K)-dependent manner. Taken together, our data reveal Asef1 functions as a negative regulator of synaptic localization of PSD-95 and synaptic transmission.

Transcription-independent expression of PKMζ in the anterior cingulate cortex contributes to chronically maintained neuropathic pain.

Protein kinase M ζ is well known for its role in maintaining memory and pain. Previously, we revealed that the activation of protein kinase M ζ in the anterior cingulate cortex plays a role in sustaining neuropathic pain. However, the mechanism by which protein kinase M ζ is expressed in the anterior cingulate cortex by peripheral nerve injury, and whether blocking of protein kinase M ζ using its inhibitor, zeta inhibitory peptide, produces analgesic effects in neuropathic pain maintained chronically after injury, have not previously been resolved. In this study, we show that protein kinase M ζ expression in the anterior cingulate cortex is enhanced by peripheral nerve injury in a transcription-independent manner. We also reveal that the inhibition of protein kinase M ζ through zeta inhibitory peptide treatment is enough to reduce mechanical allodynia responses in mice with one-month-old nerve injuries. However, the zeta inhibitory peptide treatment was only effective for a limited time.

The Brain-Enriched MicroRNA miR-9-3p Regulates Synaptic Plasticity and Memory.

UNLABELLED: MicroRNAs (miRNAs) are small, noncoding RNAs that posttranscriptionally regulate gene expression in many tissues. Although a number of brain-enriched miRNAs have been identified, only a few specific miRNAs have been revealed as critical regulators of synaptic plasticity, learning, and memory. miR-9-5p/3p are brain-enriched miRNAs known to regulate development and their changes have been implicated in several neurological disorders, yet their role in mature neurons in mice is largely unknown. Here, we report that inhibition of miR-9-3p, but not miR-9-5p, impaired hippocampal long-term potentiation (LTP) without affecting basal synaptic transmission. Moreover, inhibition of miR-9-3p in the hippocampus resulted in learning and memory deficits. Furthermore, miR-9-3p inhibition increased the expression of the LTP-related genes Dmd and SAP97, the expression levels of which are negatively correlated with LTP. These results suggest that miR-9-3p-mediated gene regulation plays important roles in synaptic plasticity and hippocampus-dependent memory. SIGNIFICANCE STATEMENT: Despite the abundant expression of the brain-specific microRNA miR-9-5p/3p in both proliferating and postmitotic neurons, most functional studies have focused on their role in neuronal development. Here, we examined the role of miR-9-5p/3p in adult brain and found that miR-9-3p, but not miR-9-5p, has a critical role in hippocampal synaptic plasticity and memory. Moreover, we identified in vivo binding targets of miR-9-3p that are involved in the regulation of long-term potentiation. Our study provides the very first evidence for the critical role of miR-9-3p in synaptic plasticity and memory in the adult mouse.

Piconewton-Scale Analysis of Ras-BRaf Signal Transduction with Single-Molecule Force Spectroscopy.

Intermolecular interactions dominate the behavior of signal transduction in various physiological and pathological cell processes, yet assessing these interactions remains a challenging task. Here, this study reports a single-molecule force spectroscopic method that enables functional delineation of two interaction sites (≈35 pN and ≈90 pN) between signaling effectors Ras and BRaf in the canonical mitogen-activated protein kinase (MAPK) pathway. This analysis reveals mutations on BRaf at Q257 and A246, two sites frequently linked to cardio-faciocutaneous syndrome, result in ≈10-30 pN alterations in RasBRaf intermolecular binding force. The magnitude of changes in RasBRaf binding force correlates with the size of alterations in protein affinity and in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-sensitive glutamate receptor (-R)-mediated synaptic transmission in neurons expressing replacement BRaf mutants, and predicts the extent of learning impairments in animals expressing replacement BRaf mutants. These results establish single-molecule force spectroscopy as an effective platform for evaluating the piconewton-level interaction of signaling molecules and predicting the behavior outcome of signal transduction.

Activation of Aplysia ARF6 induces neurite outgrowth and is sequestered by the overexpression of the PH domain of Aplysia Sec7 proteins.

ADP-ribosylation factors (ARFs) are small guanosine triphosphatases of the Ras superfamily involved in membrane trafficking and regulation of the actin cytoskeleton. Aplysia Sec7 protein (ApSec7), a guanine nucleotide exchange factor for ARF1 and ARF6, induces neurite outgrowth and plays a key role in 5-hydroxyltryptamine-induced neurite growth and synaptic facilitation in Aplysia sensory-motor synapses. However, the specific role of ARF6 signaling on neurite outgrowth in Aplysia neurons has not been examined. In the present study, we cloned Aplysia ARF6 (ApARF6) and revealed that an overexpression of enhanced green fluorescent protein (EGFP)-fused constitutively active ApARF6 (ApARF6-Q67L-EGFP) could induce neurite outgrowth in Aplysia sensory neurons. Further, we observed that ApARF6-induced neurite outgrowth was inhibited by the co-expression of a Sec7 activity-deficient mutant of ApSec7 (ApSec7-E159K). The pleckstrin homology domain of ApSec7 may bind to active ApARF6 at the plasma membrane and prevent active ApARF6-induced functions, including intracellular vacuole formation in HEK293T cells. The results of the present study suggest that activation of ARF6 signaling could induce neurite outgrowth in Aplysia neurons and may be involved in downstream signaling of ApSec7-induced neurite outgrowth in Aplysia neurons.

Dual roles of the N-terminal coiled-coil domain of an Aplysia sec7 protein: homodimer formation and nuclear export.

Cytohesin family proteins act as guanine nucleotide exchange factors (GEFs) for the ADP-ribosylation factor family of small GTP-binding proteins. Aplysia Sec7 (ApSec7), a member of the cytohesin family in Aplysia, plays key roles in neurite outgrowth in Aplysia neurons. Although ApSec7 has a conserved coiled-coil (CC) domain, its role was not clear. In this study, we found that the CC domain of ApSec7 and ARNO/cytohesin 2 are involved in homodimer formation, leading to efficient plasma membrane targeting of ApSec7 and ARNO/cytohesin 2 in HEK293T cells. Therefore, deletion of the CC domain of ApSec7 and ARNO/cytohesin 2 may result in a loss of dimerization and reduce plasma membrane localization. In addition, the CC domains of ApSec7 and ARNO/cytohesin 2 have partially or fully CRM1-dependent nuclear export signals, respectively. Taken together, our results suggest that the CC domain of cytohesin family proteins, including ApSec7 and ARNO/cytohesin 2, has dual roles in intracellular targeting: increased plasma membrane targeting through homodimer formation and nuclear exclusion through either a CRM1-dependent or a CRM1-independent pathway.

Neuronal mechanisms and circuits underlying repetitive behaviors in mouse models of autism spectrum disorder.

Autism spectrum disorder (ASD) refers to a broad spectrum of neurodevelopmental disorders characterized by three central behavioral symptoms: impaired social interaction, impaired social communication, and restricted and repetitive behaviors. However, the symptoms are heterogeneous among patients and a number of ASD mouse models have been generated containing mutations that mimic the mutations found in human patients with ASD. Each mouse model was found to display a unique set of repetitive behaviors. In this review, we summarize the repetitive behaviors of the ASD mouse models and variations found in their neural mechanisms including molecular and electrophysiological features. We also propose potential neuronal mechanisms underlying these repetitive behaviors, focusing on the role of the cortico-basal ganglia-thalamic circuits and brain regions associated with both social and repetitive behaviors. Further understanding of molecular and circuitry mechanisms of the repetitive behaviors associated with ASD is necessary to aid the development of effective treatments for these disorders.

Intracellular membrane association of the Aplysia cAMP phosphodiesterase long and short forms via different targeting mechanisms.

Phosphodiesterases (PDEs) play key roles in cAMP compartmentalization, which is required for intracellular signaling processes, through specific subcellular targeting. Previously, we showed that the long and short forms of Aplysia PDE4 (ApPDE4), which are localized to the membranes of distinct subcellular organelles, play key roles in 5-hydroxytryptamine-induced synaptic facilitation in Aplysia sensory and motor synapses. However, the molecular mechanism of the isoform-specific distinct membrane targeting was not clear. In this study, we further investigated the molecular mechanism of the membrane targeting of the ApPDE4 long and short forms. We found that the membrane targeting of the long form was mediated by hydrophobic interactions, mainly via 16 amino acids at the N-terminal region, whereas the short form was targeted solely to the plasma membrane, mainly by nonspecific electrostatic interactions between their N termini and the negatively charged lipids such as the phosphatidylinositol polyphosphates PI4P and PI(4,5)P2, which are embedded in the inner leaflet of the plasma membrane. Moreover, oligomerization of the long or short form by interaction of their respective upstream conserved region domains, UCR1 and UCR2, enhanced their plasma membrane targeting. These results suggest that the long and short forms of ApPDE4 are distinctly targeted to intracellular membranes through their direct association with the membranes via hydrophobic and electrostatic interactions, respectively.

Pain perception in acute model mice of Parkinson's disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

BACKGROUND: Pain is the most prominent non-motor symptom observed in patients with Parkinson's disease (PD). However, the mechanisms underlying the generation of pain in PD have not been well studied. We used a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD to analyze the relationship between pain sensory abnormalities and the degeneration of dopaminergic neurons. RESULTS: The latency to fall off the rotarod and the total distance traveled in round chamber were significantly reduced in MPTP-induced PD mice, consistent with motor dysfunction. MPTP-treated mice also showed remarkably shorter nociceptive response latencies compared to saline-treated mice and the subcutaneous injection of L-3,4-dihydroxyphenylalanine (L-DOPA) partially reversed pain hypersensitivity induced by MPTP treatment. We found that degeneration of cell bodies and fibers in the substantia nigra pars compacta and the striatum of MPTP-treated mice. In addition, astrocytic and microglial activation was seen in the subthalamic nucleus and neuronal activity was significantly increased in the striatum and globus pallidus. However, we did not observe any changes in neurons, astrocytes, and microglia of both the dorsal and ventral horns in the spinal cord after MPTP treatment. CONCLUSIONS: These results suggest that the dopaminergic nigrostriatal pathway may have a role in inhibiting noxious stimuli, and that abnormal inflammatory responses and neural activity in basal ganglia is correlated to pain processing in PD induced by MPTP treatment.

Understanding the Modulatory Effects of Cannabidiol on Alzheimer's Disease.

Alzheimer's disease (AD), the most common neurodegenerative disease, is characterized by progressive cognitive impairment. The deposition of amyloid beta (Aß) and hyperphosphorylated tau is considered the hallmark of AD pathology. Many therapeutic approaches such as Food and Drug Administration-approved cholinesterase inhibitors and N-methyl-D-aspartate receptor antagonists have been used to intervene in AD pathology. However, current therapies only provide limited symptomatic relief and are ineffective in preventing AD progression. Cannabidiol (CBD), a phytocannabinoid devoid of psychoactive responses, provides neuroprotective effects through both cannabinoid and noncannabinoid receptors. Recent studies using an AD mouse model have suggested that CBD can reverse cognitive deficits along with Aß-induced neuroinflammatory, oxidative responses, and neuronal death. Furthermore, CBD can reduce the accumulation of Aß and hyperphosphorylation of tau, suggesting the possibility of delaying AD progression. Particularly, the noncannabinoid receptor, peroxisome proliferator-activated receptor gamma, has been suggested to be involved in multiple functions of CBD. Therefore, understanding the underlying mechanisms of CBD is necessary for intervening in AD pathology in depth and for the translation of preclinical studies into clinical settings. In this review, we summarize recent studies on the effect of CBD in AD and suggest problems to be overcome for the therapeutic use of CBD.

Perseverative stereotypic behavior of Epac2 KO mice in a reward-based decision making task.

Successfully navigating dynamic environments requires balancing the decision to stay at an optimal choice with that to switch to an alternative to acquire new knowledge. However, the genetic factors and cellular activity shaping this "stay or switch" action decision remains largely unidentified. Here we find that mice carrying a deletion of the exchange protein directly activated by cAMP 2 (Epac2) gene, a putative autism locus, exhibit perseverative "stay" behavior in a dynamic foraging task. Anatomical analysis found that the loss of Epac2 resulted in a significant decrease in the density of PV-expressing interneurons in the ventrolateral orbitofrontal cortex (OFC) and dorsal striatum (dSTR). Further, in vitro whole cell patch clamp recordings of PV+ GABAergic interneurons in the dSTR revealed altered neural activity in Epac2 KO mice in response to dopamine. Our findings highlight a potential role of Epac2 in structural changes and neural responses of PV-expressing GABAergic interneurons in the ventrolateral OFC and dSTR during value-based reinforcement learning and link Epac2 function to abnormal decision-making processes and perseverative behaviors seen in autism.

Identification of a novel Shank2 transcriptional variant in Shank2 knockout mouse model of autism spectrum disorder.

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders that are highly heterogeneous in clinical symptoms as well as etiologies. Mutations in SHANK2 are associated with ASD and accordingly, Shank2 knockout mouse shows ASD-like behavioral phenotypes, including social deficits. Intriguingly, two lines of Shank2 knockout (KO) mouse generated by deleting different exons (exon 6-7 or exon 7) showed distinct cellular phenotypes. Previously, we compared gene expressions between Shank2 KOs lacking exon 6-7 (e6-7 KO) and KOs lacking exon 7 (e7 KO) by performing RNA-seq. In this study, we expanded transcriptomic analyses to identify novel transcriptional variants in the KO mice. We found prominent expression of a novel exon (exon 4' or e4') between the existing exons 4 and 5 in the Shank2 e6-7 KO model. Expression of the transcriptional variant harboring this novel exon was confirmed by RT-PCR and western blotting. These findings suggest that the novel variant may function as a modifier gene, which contributes to the differences between the two Shank2 mutant lines. Furthermore, our result further represents an example of genetic compensation that may lead to phenotypic heterogeneity among ASD patients with mutations in the same gene.

Autophagy pathway upregulation in a human iPSC-derived neuronal model of Cohen syndrome with VPS13B missense mutations.

Significant clinical symptoms of Cohen syndrome (CS), a rare autosomal recessive disorder, include intellectual disability, facial dysmorphism, postnatal microcephaly, retinal dystrophy, and intermittent neutropenia. CS has been associated with mutations in the VPS13B (vacuolar protein sorting 13 homolog B) gene, which regulates vesicle-mediated protein sorting and transport; however, the cellular mechanism underlying CS pathogenesis in patient-derived neurons remains uncertain. This report states that autophagic vacuoles accumulate in CS fibroblasts and the axonal terminals of CS patient-specific induced pluripotent stem cells (CS iPSC)-derived neurons; additionally, autophagic flux was significantly increased in CS-derived neurons compared to control neurons. VPS13B knockout HeLa cell lines generated using the CRISPR/Cas9 genome editing system showed significant upregulation of autophagic flux, indicating that VSP13B may be associated with autophagy in CS. Transcriptomic analysis focusing on the autophagy pathway revealed that genes associated with autophagosome organization were dysregulated in CS-derived neurons. ATG4C is a mammalian ATG4 paralog and a crucial regulatory component of the autophagosome biogenesis/recycling pathway. ATG4C was significantly upregulated in CS-derived neurons, indicating that autophagy is upregulated in CS neurons. The autophagy pathway in CS neurons may be associated with the pathophysiology exhibited in the neural network of CS patients.

Cohen Syndrome Patient iPSC-Derived Neurospheres and Forebrain-Like Glutamatergic Neurons Reveal Reduced Proliferation of Neural Progenitor Cells and Altered Expression of Synapse Genes.

Cohen syndrome (CS), a rare autosomal recessive disorder, has been associated with genetic mutations in the VPS13B gene, which regulates vesicle-mediated protein sorting and transport. However, the cellular mechanism underlying CS pathogenesis in patient-derived human neurons remains unknown. We identified a novel compound heterozygous mutation, due to homozygous variation of biparental origin and heterozygous variation inherited from the father, in the VPS13B gene in a 20-month-old female patient. To understand the cellular pathogenic mechanisms, we generated induced pluripotent stem cells (iPSCs) from the fibroblasts of the CS patient. The iPSCs were differentiated into forebrain-like functional glutamatergic neurons or neurospheres. Functional annotation from transcriptomic analysis using CS iPSC-derived neurons revealed that synapse-related functions were enriched among the upregulated and downregulated genes in the CS neurons, whereas processes associated with neurodevelopment were enriched in the downregulated genes. The developing CS neurospheres were small in size compared to control neurospheres, likely due to the reduced proliferation of SOX2-positive neural stem cells. Moreover, the number of SV2B-positive puncta and spine-like structures was significantly reduced in the CS neurons, suggesting synaptic dysfunction. Taking these findings together, for the first time, we report a potential cellular pathogenic mechanism which reveals the alteration of neurodevelopment-related genes and the dysregulation of synaptic function in the human induced neurons differentiated from iPSCs and neurospheres of a CS patient.

Spatial Learning and Motor Deficits in Vacuolar Protein Sorting-associated Protein 13b (Vps13b) Mutant Mouse.

Vacuolar protein sorting-associated protein 13B (VPS13B), also known as COH1, is one of the VPS13 family members which is involved in transmembrane transport, Golgi integrity, and neuritogenesis. Mutations in the VPS13B gene are associated with Cohen syndrome and other cognitive disorders such as intellectual disabilities and autism spectrum disorder (ASD). However, the pathophysiology of VPS13B-associated cognitive deficits is unclear, in part, due to the lack of animal models. Here, we generated a Vps13b exon 2 deletion mutant mouse and analyzed the behavioral phenotypes. We found that Vps13b mutant mice showed reduced activity in open field test and significantly shorter latency to fall in the rotarod test, suggesting that the mutants have motor deficits. In addition, we found that Vps13b mutant mice showed deficits in spatial learning in the hidden platform version of the Morris water maze. The Vps13b mutant mice were normal in other behaviors such as anxiety-like behaviors, working memory and social behaviors. Our results suggest that Vps13b mutant mice may recapitulate key clinical symptoms in Cohen syndrome such as intellectual disability and hypotonia. Vps13b mutant mice may serve as a useful model to investigate the pathophysiology of Vps13b-associated disorders.